Abstract

Plamid constructs pNW1 through pNW6 containing a controllable xylE gene (for catechol 2,3-dioxygenase) were introduced into Streptomyces lividans strains to provide a selectable marker system. xylE functions in S. lividans under the control of bacteriophage lambda promoters lambda-p(L) and lambda-p(R). Thermoregulated expression of xylE is provided through the lambda repressor cI857. Catechol 2,3-dioxygenase activity was increased 2.8-fold from plasmid construct pNW2 (lambda-p(L), xylE, cI857) and 9.5- and 7.4-fold from constructs pNW3 (lambda-p(R), xylE, cI857) and pNW5 (lambda-p(R), xylE, cI857), respectively, when the temperature was shifted from 28-degrees-C to 37-degrees-C. The stability of the constructs varied from 4.7% for pNW2 to 99.4% for pNW4 (lambda-p(L), xylE) over two rounds of sporulation. Marked S. lividans strains released into soil systems retained the XylE phenotype for more than 80 days, depending on the marker plasmid, when examined by a selective plating method. Furthermore, S. lividans harboring plasmid pNW5 was detectable by nucleic acid hybridization at less than 10 CFU g-1 (dry weight) of soil as mycelium and 10(3) CFU g-1 (dry weight) of soil as spores with the xylE marker DNA extracted from soil and amplified by using the polymerase chain reaction.